Isolation, culture, and transfection of primary mammary epithelial organoids Primary mammary epithelial organoids were prepared from 8-week-old CD1 mice (Charles

Supplementary Experimental Procedures Isolation, culture, and transfection of primary mammary epithelial organoids Primary mammary epithelial organoids were prepared from 8-week-old CD1 mice (Charles River) as previously described (Nelson et al., 2006). Inguinal glands were removed aseptically, minced with razor blades, and incubated with agitation (350 rpm) at 37°C in DMEM/F12 supplemented with 0.2% collagenase (Roche), 0.2% trypsin (Sigma), 5% FBS, 5 μg/ml insulin, and 50 μg/ml gentamicin. The digested tissue was centrifuged at 1800 rpm for 10 min. The supernatant was removed, centrifuged three times, and the resulting cell pellet was resuspended in DMEM/F12 supplemented with 20 U/ml DNase I (Sigma). The organoids were separated from single cells (mainly fibroblasts) using differential centrifugation. Organoids were resuspended in DMEM/F12 supplemented with 10% FBS, 5 ng/ml EGF, insulin/transferrin/sodium selenite (ITS; Sigma), penicillin/streptomycin (Sigma), and gentamicin, and immediately embedded in collagen gels or plated on collagen-coated plastic for transfection.